基因沉默Delta N p63对膀胱肿瘤生长抑制和CDK4的影响

Inhibitory effect of tumor growth and CKD4 expressing by silencing Delta N p63 gene on human transitional cell carcinoma of bladder

目的采用RNA干扰(RNAi)方法,研究当Delta N p63基因的表达沉默后,对人膀胱癌裸鼠皮下移植模型肿瘤的抑制作用,并检测对细胞周期素依赖性激酶4(CDK4)基因和蛋白表达的影响,进一步揭示Delta N p63基因在膀胱肿瘤中的作用机制。方法首先构建Delta N p63基因特异性短发卡RNA(shRNA)表达质粒,将表达质粒转染至裸鼠皮下移植模型肿瘤瘤体内,比较各组肿瘤体积,光镜下检测肿瘤组织病理学改变,RT-PCR方法检测CKD4基因的表达变化,Western blot和SP免疫组化检测CKD4蛋白表达变化。结果质粒注射到肿瘤8周后,治疗组、对照组与生理盐水组比较,治疗组与生理盐水组瘤体积比较差异有统计学意义(P<0.01),抑瘤率74.62%。光镜结果显示治疗组肿瘤生长受到明显抑制,细胞核分裂象明显减少,可见坏死和凋亡肿瘤细胞、局部炎症细胞浸润。RT-PCR结果显示治疗组CKD4基因表达降低,蛋白检测结果显示CKD4蛋白表达下降。结论沉默Delta N p63表达后,可显著抑制人膀胱癌裸鼠肿瘤模型中肿瘤的生长,Delta N p63可能通过抑制CKD4的表达而抑制肿瘤的细胞生长。

Objective To explore the inhibitory effect of tumor growth and ckd4 expressing by silencing Delta N p63 gene with short hairpin RNA on human transitional cell carcinoma of the bladder（TCCB） in xenograft nude mice through RNAi technique.Methods Human TCCB 5637 cells were transplanted into nude mice to establish xenograft tumors,Delta N p63 specific shRNA expression plasmid was transfected into the tumors,the tumor volume was observed,tumor morphology was observed with HE staining.The inhibitory effect of ckd4 gene was examined with semi-quantitative reverse transcriptase-polymerase chain reaction（RT-PCR）,ckd4 protein was examined by the immunohistochemical staining assay and Western blot.Results Delta N p63 specific shRNA expression plasmid was transfected into the tumors.After 8 weeks,the tumor volume was significantly smaller in the treated group than in the control group and the normal saline group（P0.01）,the inhibition rate was 74.62% in the treated group.In the treated group the necrosis and decrease of karyokinesis were found under light microscope,inflammatory cells were also found,there was no such change in the control group and the normal saline group.Delta N p63-shRNA directly down-regulated the expression of ckd4 protein and mRNA.Conclusion The Delta N p63-shRNA expression plasmid can significantly inhibit the growth of TCCB in nude mice and also can inhibit the cellular proliferation of TCCB through inhibit ckd4.